plants
Article
Micromorphology and Histology of the Secretory Apparatus of
Diospyros villosa (L.) de Winter Leaves and Stem Bark
Oluwatosin Temilade Adu 1 , Yougasphree Naidoo 1 , Temitope Samson Adu 2 , Venkataramegowda Sivaram 3 ,
Yaser Hassan Dewir 4, * and Hail Rihan 5,6
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2
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4
5
6
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Citation: Adu, O.T.; Naidoo, Y.; Adu,
T.S.; Sivaram, V.; Dewir, Y.H.; Rihan,
H. Micromorphology and Histology
of the Secretory Apparatus of
Diospyros villosa (L.) de Winter Leaves
and Stem Bark. Plants 2022, 11, 2498.
https://doi.org/10.3390/
plants11192498
Academic Editors: Claudia Giuliani
and Fico Gelsomina
Received: 2 August 2022
Accepted: 20 September 2022
Published: 23 September 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affiliations.
School of Life Sciences, Westville Campus, University of KwaZulu-Natal, Private Bag X54001,
Durban 4000, South Africa
Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife 220282, Nigeria
Laboratory of Biodiversity and Apiculture, Department of Botany, Bangalore University,
Bangalore 560056, India
Plant Production Department, College of Food and Agriculture Sciences, King Saud University,
Riyadh 11451, Saudi Arabia
School of Biological Sciences, Faculty of Science and Environment, University of Plymouth,
Plymouth PL4 8AA, UK
Phytome Life Sciences, Launceston PL15 7AB, UK
Correspondence: ydewir@ksu.edu.sa
Abstract: Diospyros villosa is a perennial species prominently acknowledged for its local medicinal
applications. The native utilisation of this species in traditional medicine may be ascribed to the
presence of secretory structures and their exudate (comprised of phytochemicals). However, the
morphological nature and optical features of the secretory structures in D. villosa remain largely
unclear. This study was directed to ascertain the occurrence and adaptive features of structures
found within the leaves and stem bark of D. villosa using light and electron microscopy techniques.
The current study notes the existence of trichomes, and other secretory structures were noted. SEM
indicated the presence of non-glandular hirsute trichomes with bulky stalk on both leaves and
stem surfaces. Transverse stem sections revealed the existence of crystal idioblasts. Moreover, the
presence of the main phytochemical groups and their localisation within the foliage and stem bark
was elucidated through various histochemical tests. The trichomal length and density were also
assessed in leaves at different stages of development. The results indicated that the trichomal density
at different stages of development of the D. villosa leaves and stem bark was not significantly different
from one another, F(3,39) = 1.183, p = 0.3297. The average length of the non-glandular trichomes in
the emergent, young and mature leaves, as well as in the stem, was recorded to be 230 ± 30.6 µm,
246 ± 40.32 µm, 193 ± 27.55 µm and 164 ± 18.62 µm, respectively. The perimeter and circumference
of the observed trichomes in the developmental stages of D. villosa leaf and the stem bark were not
statistically different, F(3,39) = 1.092, p = 0.3615. The results of histochemical tests showed the existence
of phenols alkaloids, which are medicinally important and beneficial for treatment of diseases. The
findings of this study, being reported for the first time may be considered in establishing microscopic
and pharmacognostic measure for future identification and verification of natural herbal plant.
Trichomal micromorphology and histological evaluations could be utilised as a tool for appropriate
description for the assessment of this species.
Keywords: histochemistry; microscopy; secretory; trichomes
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
The genus Diospyros is comprised of more than 350 species [1] and is considered most
important due to its high economic value [2]. Diospyros is made up of shrubs and trees
which are distributed across the world. Almost 42 species can be found in India, within
the Central Deccan Plateau dry deciduous forest, tropical dry deciduous forest at Assam
Plants 2022, 11, 2498. https://doi.org/10.3390/plants11192498
https://www.mdpi.com/journal/plants
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and Bengal Safari forest [3,4]. Furthermore, various species within the genus Diospyros are
located around Africa and are highly important in both the traditional medicine and food
industry [5]. D. villosa is a well-known species used in the maintenance of oral hygiene [6].
D. villosa is a plant that occurs naturally throughout the African continent [7]. D. villosa
is a perennial, bushy, evergreen plant which could be as tall as 1–4 m. The leaves are
chartaceous, dry-dull brown in colour upon on the abaxial surface and much paler on the
adaxial surface. The foliage is, on average, 3 cm long, 2.5 cm wide and bovate/oblong
in shape. The leaf apex is usually broadly rounded, slightly emarginated and sometimes
obtuse, whereas the base is often in cordate or round shaped [6]. D. villosa leaves are
particularly noted for having distinct hair-like epidermal structures which are referred to as
‘trichomes’. Trichomes are uni- or multi-celled structures which originate from epidermal
cells of the aerial organs. These epidermal structures vary significantly in morphological
characters, location, ability to secrete and type of secretion [8]. Trichomes have many
functional roles within a plant. These include protection to the plant from external stress or
mechanical damage [9], decreasing the heat load of a plant, maximising freezing tolerance,
participation in seed dispersal, retaining water balance in plant leaves, deflection of intense
radiation of the sun and protection against herbivores [10]. Additionally, the glandular
trichomes offer chemical protection against different plant eating microorganisms and
higher animals [11]. In addition, the exudate may be of prime importance and use in the
medicinal industry
There is a lack of scientific data on the trichome morphology for many ‘Diospyros’
species. The morphology and structure of trichomes and the exudate found in D. villosa
have been scarcely studied. The primary focus of this study was to investigate the histomorphology and histochemistry of the leaves and stem bark of D. villosa. The nature of
secretory products in the leaves and stem bark were also investigated using histochemical
assays in order to assess the existing constituents of the plant.
2. Materials and Methods
2.1. Plant Collection
Freshly harvested foliage and stem bark material of D. villosa were collected from
KwaZulu-Natal, Durban, South Africa (29◦ 84′ 33.6′′ S, 31◦ 4′ 12′′ E). The plant was identified
and deposited in the herbarium with number (01/18257) at the School of Life Sciences,
University of KwaZulu-Natal, Durban. These samples were utilised for histological staining
and morphological assessment. The developmental stages of the leaf were categorised as
emergent, young and mature. A total number of ten replicates were made for each stage of
the leaves and stem bark.
2.2. Stereomicroscopy
The structures at the top surface (abaxial) and underneath (adaxial) of the leaf and stem
bark of the plant material were observed with an AZ-LED ring furnished stereomicroscope.
Images were captured and processed with a Nikon AZ100 stereomicroscope furnished with
a camera and the Nikon NISD Elements Software (Version 3.00, Nikon, Tokyo, Japan).
2.3. Electron Microscopy
Electron microscopy was used to examine the trichomal morphology in the leaf and
stem of D. villosa. The samples were studied systemically with the aid of Nikon AZ100
stereomicroscope, Japan, attached to Nikon Fibre Illuminator and images were taken using
a Nikon DXM1200C digital camera. The images were taken using the NIS Element Software.
2.4. Scanning Electron Microscopy
The dirt observed to have blocked the surface of the samples was washed off so as to
reduce the ubiquity. Then, the leaves samples belonging to each developmental stage and
the stem sections were washed with distilled water and, subsequently, with few drops of
Bio-Rad tween-20 solution. The leaves and stem sections were washed once with distilled
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water. The sections were sectioned and fixed using glutaraldehyde (2.5%) in phosphate
buffer (0.1 M, pH 7.2) for 24 h. The samples were preserved and made stable by allowing
buffer washes thrice for 5 min and fixed in osmium tetroxide (0.5%) for 2 h in the absence of
sunlight. Samples were later dehydrated in serial solutions (i.e., 25%, 50%, 70% and 100%).
The Quorum K180 critical point dryer was used to dry the samples and later placed on
aluminium stubs with the aid of carbon conductive tape and sputter-coated with gold in a
QurumQ150 RES gold coater. The samples were viewed with LEO 1450 SEM (SmartSEM)
and images were captured and analysed.
2.5. Transmission Electron Microscopy
Segments of leaf and stem tissues were excised and fixed in glutaraldehyde (2.5%) in
phosphate buffer (0.1 M, pH 7.2) for 24 h. These sections were later washed thrice with
buffer and fixed in osmium tetroxide (0.5%) in the absence of sunlight for 2 h. Following
this, the sections were dehydrated in graded doses of acetone, i.e., 25%, 50%, 70% and
100%. The sections were infiltrated with 50% propylene oxide (50%) and Spurr’s resin for
24 h. This was further followed by allowing polymerisation of sections in 100% resin at
a temperature of 85 ◦ C for 8 h. The resin blocks were sectioned using the LKB 7801A on
a Leica EM UC7 microtome (Leica Microsystems, Germany). The thinner sections were
collected and stained in uranyl acetate (2.5%) for 10 min. The sections were further rinsed
with slightly warm water and stained with 2.5% lead citrate and finally rinsed before
viewing under a Jeol 1010 transmission electron microscope.
2.6. Light Microscopy
The sections from leaf and stem bark were obtained as described in the procedure
for transmission electron microscopy. The Leica ultramicrotome EM UC7 (Leica Microsystems, Germany) was used for the sectioning and the sections were then stained with 1%
toluidine blue for 1 min. The stained sections were viewed under a Nikon eclipse, 80i
light microscope.
2.7. Histochemistry
The excision of sections with 100 µm thickness was made possible with the help
of dental wax while using an Oxford vibratome. The obtained sections were hydrated
and subsequently stained accordingly. The sections were stained with toluidine blue to
detect carboxylated polysaccharides [12]. The sections were also stained with mercuric
bromophenol blue to indicate total proteins [13]. Sudan back as well as Sudan IV was
further used for the confirmation of total lipids and fatty acids [14]. The confirmatory
test for the presence of phenolic compound was conducted using ferric trichloride [15].
The ruthenium red was used for the detection of acidic polysaccharides [14]. Meanwhile,
the confirmatory test for the presence of alkaloids was performed using Wagner’s and
Dittmar’s reagents [16].
2.8. Fluorescence Microscopy
Fresh hand cut sections of the leaf and stem bark were utilised for the purpose of
this assay. The sections were mounted on the glass slide and viewed. The images were
captured at various wavelengths (300 nm, 330 nm and 380 nm) using the Zeiss (Oberkochen,
Germany) LSM 710 microscope, Germany. The stem bark sections were stained with 2%
acridine orange for a period of 2 min. The sections were later rinsed with distilled water.
The prepared sections were placed on the Zeiss LSM 710 microscope and images were
captured at 488 nm. Furthermore, the obtainable leaf and stem sections were stained with
Calcofluor White for 2 min and rinsed using distilled water. The sections were further
placed in water and viewed using an epifluorescence microscope (Nikon Eclipse ATI) at a
wavelength of 365 nm. The histomorphology of the embedded structures in the plant was
conducted with the aid of Calcofluor White [17]. This stain may stain callose rather than
being attached to the cellulose.
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2.9. Energy Dispersive X-ray Microanalysis (EDX)
The elemental constituents of the leaves at developmental stages, as well as the stem
bark of D. villosa, were determined. The elemental composition and quantification of both
leaves and stem were detected by Oxford EDX detector (Oxford Instruments, Oxfordshire,
UK) in a set-up of Zeiss Ultra Plus FEG-SEM (Oberkochen, Germany) at 20 kV [18]. The
leaf area analyses were conducted to determine the chemical constituents of the secretory
products from the plant.
2.10. Trichome Density, Length and Statistical Analysis
A good choice of images acquired from SEM was analysed using Image J software.
The statistical package GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA)
was used for the data analysis. The trichomes on the leaves and stem surfaces were
counted. Likewise, the trichomal density, average length, perimeter and circumference
were analysed using Image J software. The quantified observations were further analysed
using one-way analysis of variance (one-way ANOVA) and a Bonferroni test was used as the
post-hoc analysis. The normality of data was assessed by Kolmogrov–Smirnov test and the
acquired data were compared with one another using one way ANOVA when the expected
requirements are duly met. p values less than 0.05 was standard as being significant.
3. Results
3.1. Stereomicroscopy
Stereomicrographs revealed that the abaxial surface of the leaf was predominantly
occupied by trichomes compared with the adaxial surface (Figure 1a,b). Stereomicrographs
of D. villosa revealed the sole type of trichomes (non-glandular), which was found on the
stem bark (Figure 2a–d). The transverse section of D. villosa stem further revealed the
presence of crystal idioblasts (Figure 2d).
Figure 1. Stereomicrograph indicating the leaf topology of D. villosa. (a) Abaxial surface of the leaf
with dense non-glandular trichomes along the mid and lateral veins. (b) Adaxial surface of the leaf
showing fewer non-glandular trichomes. NG = Non glandular trichomes.
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Figure 2. SEM micrograph of the leaf and stem bark of D. villosa. (a) Abaxial leaf surface showing
the presence of numerous non-glandular trichomes across the leaf surface. (b) Adaxial leaf surface
showing scanty non-glandular trichome coverage. (c) Stem surface indicating the distribution of
non-glandular trichomes and (d) transverse sectional area of the stem bark. NG = Non glandular,
MV = Medial vein, St = Stalk/base of the trichome, Cr = Crystal.
3.2. Scanning Electron Microscopy
Scanning electron micrographs of the leaves showed secretory pores on the epidermis
(Figure 3a). Stomata/secretory pores appeared in abundance on the mature leaves’ adaxial
surface compared to both the emergent and young leaves. Micrographs further revealed a
single non-glandular trichome type (Figure 3c,d).
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Figure 3. SEM of D. villosa leaves showing: (a) stomata (S) on mature leaf adaxial surface and
(b) non-glandular single-celled stalk on the adaxial surface of mature leaf (St). SEM of D. villosa stem
showing (c) non-glandular hirsute (U) trichome on surface of the stem bark and (d) non glandular
trichome with bulky stalk (St).
3.3. Transmission Electron Microscopy (TEM)
The D. villosa leaves and stem bark sections were assessed using TEM. There was
further observation of different molecular components, such as endoplasmic reticulum,
vesicles, vacuoles (large), mitochondria, starch granule, ribosome, chloroplast and nuclei
(Figure 4). The leaf was observed to consist of large vacuoles and a nucleus (Figure 4a).
Moreover, plasmodesmatal connection was observed in the leaf (Figure 4b). Cytoplasm
containing numerous plastids (Figure 4a) and ribosomes was also seen. Plastids were also
observed to have lipophilic material, which was indicated by the presence of dark black
deposits within. Furthermore, cytoplasm was seen to contain dense materials. The presence
of a complex network of endoplasmic reticulum, mitochondria and plastids (Figure 4c) was
further observed in the leaves. These organelles were mostly large and abundant in the
cytoplasm. The vacuoles, chloroplast and vesicles were found sufficiently along the cell
wall periphery (Figure 4c). Similarly, the stem was observed to be filled with large vacuoles
and well observed chloroplasts (Figure 5a,b).
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Figure 4. TEM micrograph of the D. villosa leaves at different developmental stages (a) Mature,
(b) Young and (c) Emergent. Ch = Chloroplast, Pl = Plastids, San DiegoP = Plasmodesmata,
Mt = Mitochondria, V = Vacuole, Nu = Nucleus, SG = Starch granules, ER = Endoplasmic Reticulum and R = Ribosome.
Figure 5. TEM micrograph of the D. villosa stem (a,b). Ch = Chloroplast, V = Vacuole, Nu = Nucleus.
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3.4. Trichome Density, Length, Perimeter and Circumference
Trichome density appeared to be similar across all developmental stages of the
D. villosa leaf. Although the trichome density observed on the stem bark appeared to be
higher compared to the leaf surfaces (Figure 6a), the ANOVA indicated that there were no
statistical difference in the trichomal density among the developmental stages of D. villosa
leaf and stem bark, F(3,39) = 1.183, p = 0.3297. Similarly, the average lengths of the nonglandular trichomes were approximately 230 ± 30.6 µm, 246 ± 40.32 µm, 193 ± 27.55 µm
and 164 ± 18.62 µm (Figure 6b). One-way ANOVA further showed that there was no
significant difference in the average trichomal length, F(3,39) = 1.478, p = 0.2369. The perimeter and circumference of the trichomes in the developmental stages of D. villosa leaf and
the stem bark were not statistically different, F(3,39) = 1.092, p = 0.3615 and F(3,39) = 0.2717,
p = 0.8454 (Figure 6c,d), respectively.
Figure 6. Density (a), average length (b), perimeter (c) and circumference (d) of non-glandular
trichomes at different developmental stage of D. villosa leaves and stem bark.
3.5. Histochemistry
The histochemical analysis showed that lipids, phenols and alkaloids were present in
the leaves and stem bark (Figures 7 and 8). The greenish-brown colour further implied that
the phenolic compounds (Figure 7a,b), the brown colouration as indicated in Figure 8c,d
confirmed the presence of alkaloids and the black deposit indicated the presence of lipids
(Figure 7e,f). These results agreed with the histochemical tests whereby various reactions
indicated different colourations indicating the presence of different compounds as shown
in Table 1.
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Figure 7. Light micrographs showing the histochemical staining characterisation of both leaf and
stem sections of D. villosa. (a) Phenolic compounds stained brown on the leaf surface with Toluidine.
(b) Phenolic compounds stained greenish brown in the stem with Toluidine. (c) Alkaloids stained
brown colour on the D. villosa leaf with Dittmar reagent. (d) Alkaloids stained brown on D. villosa
stem with Dittmar reagent. (e) Lipids stained with black stains on the leaf surface with Sudan black.
(f) Lipids stained with black on the cross section of the stem with Sudan black.
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Figure 8. Light micrographs showing histochemical characterisation of trichomes of D. villosa. (a) Alkaloids compounds in non-glandular trichomes stained brown with Dittmar reagent.
(b) Phenolic compounds stained red-brown in the stem and non-glandular (NG) trichomes with
ferric trichloride. (c) Lipids stained black in the leaf and non-glandular (NG) trichomes with Nile
blue. (d) Total protein stained purple in the stem and non-glandular trichomal section with mercuric
bromophenol blue. (e) Lipids stained yellowish black in non-glandular trichomes with Sudan III
and IV. (f) Acidic polysaccharides stained purplish red in non-glandular trichomes and leaf with
Ruthenium red scale bar = 100 µm.
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Table 1. Observations of histochemical tests on fresh leaf and stem bark sections of D. villosa.
Compounds
Stains
Leaves/Stem
Trichomes
Alkaloids
Dittmar’s
+
+
Lipids
Sudan III and IV
+
+
Nile Blue
+
+
Phenols
Ferric trichloride
+
+
Acidic Polysaccharides
Ruthenium red
+
+
Total protein
Mercuric bromophenol blue
+
+
Polyphenols (lignin,
tannins)
Toluidine blue
+
+
Reactions Observed
Brownish colouration in the stem as well as
the trichomes
Cells in the leaf and stem sections stained
black, the trichomes stained black as well
Black colouration in the leaf sections and
non-glandular trichomes
Brown deposits on the cells of the leaf
sections, the non-glandular cells further
stained brown
Leaf and non-glandular trichomes stained
purplish red
Stem cells and non-glandular trichomes
stained purple
Leaf cells and non-glandular trichomes
stained brown
(+) indicates presence of compounds.
3.6. EDX
Energy Dispersive X-ray microanalysis showed that sodium and calcium are present
in the leaf sections, and the presence of sodium is observed to be higher than the calcium
(Figure 9a,b). The mature leaves displayed the highest amount of sodium and calcium
(Figure 9a), while the emergent leaves showed the lowest concentration of salts (Figure 9c).
The sodium salts were predominantly noted within the mature leaves (Figure 9a). Similarly,
the EDX spectral showed that calcium and sodium salts are present within the stem bark;
meanwhile, the existence of calcium was higher compared to that of sodium (Figure 9d).
Figure 9. EDX spectra showing the elemental composition of the secretions of D. villosa leaf at
the mature developmental stage (a), leaf at its young developmental stage (b), leaf at its emergent
developmental stage (c) and stem bark (d).
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4. Discussion
The microscopical investigation of the leaves and stem bark of D. villosa revealed that
the trichomes were unicellular, non-glandular and longitudinally elongated on the surface.
Non glandular trichome has been previously reported for D. sericea and D. hispida [19].
However, the trichome types in Diospyros species were described as having unique, simple
or bifurcated trichomes with walls often covered with longitudinally elongated warts and
secretory cells. The microscopic analysis of trichomes in this study revealed the presence of
metabolites which further served as evidence for the presence of storage cells. Trichomes
are perfect storage structures for secondary metabolites and often rupture and discharge the
compounds at the time of damage [20,21]. This typical attribute was noted for the Diospyros
species, i.e., the surface of trichome tips possess globular secreting cells as well as elongated
spindle-shaped warts [19]. The trichomes in D. villosa showed a close resemblance to
species within the genus like D. mespiliformis, D. lotus, etc., and were, thus, considered to
significantly regulate leaf transpiration intensity by enhancing water retention of the leaf
tissues at high leaf water deficit.
The discharge exudate released through the stomata and/or secretory pores provides
a defence to the leaf against herbivory grazing. Plant nitrogen is identified by an unpleasant
taste [22]. This is particularly true of alkaloids, which are shown to be present on trichomes
located on the leaves and stem of D. villosa. It may be said that the amount of exudate
produced and released by the plant is comparatively related to the frequency of stomatal
pores. Furthermore, the presence of star-shaped crystal idioblasts was observed in the stem
sections (Figure 2d). It may be suggested that the presence of crystals in the stem sections of
D. villosa may serve as a taxonomical informative character [23] and, perhaps a mechanism
by the plant to remove excess electrolyte storage within the plant [24]. The mechanism
allows identifying the crystal idioblasts which vary in shape and size. The primary function of the crystal includes promoting electrolyte homeostasis, cell support, removal of
excess ions and electrolytes [25,26]. Similarly, crystal idioblasts are acknowledged to be
toxic due to the embedding contents having irritating and proteolytic toxins [27], which
facilitate the plant’s protection against herbivores [28]. Provided that these crystals become
extremely outsized, the surrounding cytoplasmic structures may undergo lysis, which is
quite dangerous for the plant [29,30]. The existence of crystal within the D. villosa stem may
support the mechanism promoting electrolyte homeostasis in the plant.
The size of non-glandular trichomes has a significant contribution on its relative function. These trichomes serve as a mechanical impediment to pests and a defence mechanism
for the plant and act as a form of physical protection to the underlying secretory cells [31].
In this study, the non-glandular trichome type accumulated phytocompounds. Hence,
the trichomes also played roles in the mechanical and other measures of defence against
radiation and pathogens, respectively. Similar findings were reported by Beenken [19] for
Diospyros species. Therefore, it can be explained that trichome length varied among the
families of Diospyros sp. and further contributed to the significance of the family. In addition, the findings of this study supported that secretions from trichomes are associated with
storage cells. This was further substantiated by the histochemical analysis which indicated
the presence of chemical compounds in the trichomes. Trichomes are characterised not
only by their morphology, but also by their functioning (secretion, storage and mode of
release). One may conclude that the trichomes of Diospyros differ in both morphology and
physiology. EDX analyses indicated the presence of magnesium chloride in the stem bark
of D. villosa (Figure 9d).
Examination of the morphology, distribution and the phytochemistry of the secretion
associated with leaves and stem bark of a plant could further assist in elucidating possible
functions of the trichomes of this plant [8,32]. The physical attributes like density, size
and trichomal arrangement on the leaf surface possibly promote the protection against
pests and other plant damaging organisms such as the alkaloids and phenols observed to
be present in the leaves and stem bark of D. villosa. Non-glandular trichomes could also
reduce transpiration rates and curb surface leaf exposure to intense temperatures [8,32].
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The typical non-glandular trichomes observed on D. villosa leaves and stem bark would
assume these roles as these trichomes were so dense on leaves that it is quite challenging
to assess the leaf surface directly. Since D. villosa survive in dry territories, the observed
trichomes are likely to serve a functional role in the conservation of water. This is quite
similar to a study by Hameed and Hussain [33] where trichomes in plants with higher
tolerance for survival in dry region were reported to enhance water conservation.
Although the non-glandular trichomes were regarded as non-secretory, the microscopy
of the stained leaf section indicated that the viability of both basal and stalk cells of the
trichomes (Figure 3b,d). Histochemical analysis further showed that the leaves, stem bark
and trichomes of D. villosa accumulated the phytocompounds. Therefore, these structures
played significant role in the chemical defence against insect, herbivores and pathogens.
The prominent chemical compounds responsible for these functional roles are phenols and
alkaloids. These phytochemical compounds, in accordance with Soni et al. [34], were of
medicinal importance and repute. Alkaloids are nitrogenous chemical compounds which
were reported to treat different ailments and diseases like inflammation [35], oxidative stress
and inflammation [36], asthma and fever [37]. Alkaloids appeared as active metabolites
and natural repellents against insect herbivores and natural enemies [38,39]. In addition,
phenolics were classified as abundant secondary metabolites [40]. The phenolic compounds
functioned adequately against pests and pathogens. Upon the release of phenols, they
are further oxidised to quinones by polyphenol oxidase, thus facilitating the entrapment
of insect on the leaf surface. The presence of phenolic compounds within the trichomes
(Figure 8a,b) explained the insect entrapment ability of D. villosa. Moreover, phenolics
promoted the plant’s defence against pathogens and ultraviolet radiations [41]. These
compounds were abundant in all plant segments and contained potent antioxidant activities
in comparison with other curative and pharmaceutical uses and are used in aesthetic and
lumbering industry [42].
Previous reports highlighted the importance of micromorphological and histological
screening of medicinal plants, which greatly helps the researchers in the field to develop
further research studies on medicinal plants in their respective field. The findings of the
present study will be used to supplement pharmacognostical evaluations, correct identification and verification of this plant species. The present work on D. villosa will also provide
a basic knowledge to the researchers for further studies on effect of phytocompounds
detected, characterisation and significance of other special microscopic features that can be
utilised as a measure for natural medicinal plant.
It is concluded that the emanating study provided novel information regarding the
micromorphology and functions of microstructures of D. villosa leaves and stem bark.
Non-glandular trichomes were observed on both leaves and stem bark of D. villosa. The histochemical analysis further indicated the deposition of alkaloids and phenolic compounds
in the leaves and stem bark of D. villosa. These compounds are of ecological and medicinal
importance as they have a chemical defence mechanism against pathogens and are of use in
the medicinal industry in treating a range of ailments. However, secretory products should
be further evaluated and a comprehensive phytochemical screening should be conducted
so as to establish all other phytochemicals in the plants.
Author Contributions: Conceptualisation and methodology, O.T.A. and Y.N.; investigation, O.T.A.
and Y.N.; formal analysis and data curation, O.T.A. and Y.N.; writing—original draft preparation,
O.T.A. and Y.N.; writing—review and editing, T.S.A., V.S., Y.H.D. and H.R.; validation and visualisation, T.S.A., V.S., Y.H.D. and H.R. Supervision: Y.N. All authors have read and agreed to the published
version of the manuscript.
Funding: The authors acknowledge Researchers Supporting Project number (RSP-2021/375), King Saud
University, Riyadh, Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
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Data Availability Statement: Not applicable.
Acknowledgments: The authors acknowledge Researchers Supporting Project number (RSP-2021/375),
King Saud University, Riyadh, Saudi Arabia. The authors are thankful to the Microscopy and
Microanalysis unit, University of KwaZulu-Natal, Westville campus for providing research facilities
and the National Research Foundation (NRF) South Africa, for their financial support.
Conflicts of Interest: The authors declare no conflict of interest.
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